The interaction of thrombin with methylated fibrinogen resulted in the proteolytic release of modified fibrinopeptides at a slower rate than fibrinopeptides were enzymatically hydrolyzed from unmodified fibrinogen. This esterification reduced the negative charge on the fibrinogen molecule and inhibited the subsequent action of the enzyme thrombin. The citraconylation of fibrinogen, introducing increasing negative charge concurrent with the reduction of positive charge through modification of lysine residues, increased the rate of its hydrolysis by thrombin. These results support our proposed mechanism of thrombic action as being directed by a highly negative environment in the N-terminal segments of the alpha and beta chains of fibrinogen. The ultrastructure of the methylated fibrinogen via electron microscopic examination suggests that extensive methylation produces an aggregated protein system that is less highly ordered than the specific carboxyl group modification which leads to a more highly ordered polymerized modified fibrinogen with evidence of periodicity. The fibrinopeptides participate in hemostasis via a process of vasoconstriction as examined by a newly developed rat tail bleeding assay. Hydroxylated amino acid residues appear to be involved in the active site of this biological activity.